Insulin-like growth factors (IGF-I and IGF-II) are small, highly-related proteins (˜7.5 kilodaltons) which mediate anabolic, mitogenic and anti-apoptotic activities in a wide variety of cell types. These actions result from IGF interaction with and subsequent activation of the type 1 IGF receptor (IGF1R) (Sepp-Lorenzino, (1998), Baserga, 1999). A second unrelated receptor (the type 2 IGF receptor or IGF2R) has the major function of regulation of IGF-II levels by internalisation and degradation (Wang et al., 1994) and current evidence suggests that the IGF2R acts as a tumour suppressor of IGF-II-dependent tumours (Braulke, 1999).
IGFs are produced by the liver, providing circulating IGF, and are also secreted locally in most tissues. A family of 6 high-affinity IGF binding proteins (IGFBP-1 to -6) act to increase the half-life of IGFs in circulation (predominantly as the IGFBP-3·ALS·IGF complex) and also to transport IGFs to target tissues. Within target tissues IGFBPs can either enhance or inhibit IGF action. IGFBPs can inhibit the interaction of IGF by blocking binding to the IGF1R. However, under certain circumstances IGFBPs can release IGF, thereby making IGF available for binding to the IGF1R. This results in an enhancing effect on IGF action. Release mechanisms include 1) proteolysis of the IGFBPs and 2) IGFBP binding to the extracellular matrix (ECM), both of which lower their affinity for IGF. Extracellular matrix binding is also believed to assist the localisation of IGF close to the cell surface and therefore near IGF1Rs. The outcome of IGFBP action is controlled by a balance between local proteolytic activity and the binding of IGFBPs to the ECM.
Substantial evidence (in vivo and in vitro) implicates insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in cancer. Many tumour cells (including prostate and breast) secrete more IGF-II and IGFBP-2 than their normal counterparts and their serum levels commonly rise as cancers progress (Cohen et al., (1994); Thrasher et al.,(1996); Ho et al., (1997); Chan et al., 1998). IGF secreted by tumour cells binds to the Type 1 IGF receptor potentiating tumourigenesis and metastasis (DiGiovanni et al., 2000).
The proteolysis of IGFBP-2 has been detected under a number of normal and abnormal physiological conditions. For example, IGFBP-2 fragments have been detected in human milk and cleavage occurred predominantly in the linker region between the N- and C-domains and including sites at residues 168 and 180/181 of hIGFBP-2 (Ho and Baxter, 1997; Elminger et al., 1999). Proteolysed IGFBP-2 is also found in serum during pregnancy. IGFBP-2 is also cleaved by proteases produced by cancer cells (Michell et al., 1997). The specific cancer cell proteases have not been well characterised although Cathepsin D produced in vitro by prostate epithelial cells has been shown to degrade IGFBP-2 (Kanety et al., 1993; Nunn et al., 1997). Preferential proteolysis of IGFBP-2 has been demonstrated in colonic cancers (Michell et al., 1997) and neuroblastoma cells (Menouny et al., 1997). Specific cleavage sites have not been described for proteolytic products generated by cancer proteases.
Protease cleavage sites have been identified in the IGFBP-3, -4 and -5 sequences. Proteolysis is generally within the linker regions of these proteins although it can be in the C-domain. Protease resistant IGFBP-4 and IGFBP-5 (Imai et al., 1997) have been generated by mutating specific residues at cleavage sites or by deletion of some linker region residues (deletion of 121-141 of IGFBP-4 rendered it resistant to a protease in pregnancy serum (Byun et al., 2000).
IGFBP-2 binds to human fibroblast extracellular membrane preparations (Arai et al., 1996) and glycosaminoglycans (Russo et al 1997, Arai et al., 1996). There are 2 potential matrix binding sites within the IGFBP-2 sequence. Current evidence suggests that the basic region of hIGFBP-2 (residues 227-244), corresponding to residues 201-218 of hIGFBP-5, may act as a site for matrix binding (Arai et al., 1996). Using a synthetic peptide based on residues 201-218 of hIGFBP-5 (residues known to be important for matrix binding) Arai et al., 1996, inhibited IGFBP-2 binding to heparin-Sepharose. Hodgkinson et al., (1994) predicted a glycosaminoglycan (GAG) binding site in IGFBP-2 based on a short GAG-binding consensus sequence described by Cardin and Weintraub (1989). This XBBXBX (B=basic, X=undefined) motif at residues 179-184 of hIGFBP-2 lies in the central domain. There is no published evidence that this motif plays a role in GAG binding.
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